Investigation of proto-oncogene function in normal development as opposed to tumorigenesis is now possible by using embryonic stem cell technology to examine the results of mutation in specific oncogenes in a physiological context. Gene targeting in embryonic stem cells has been used to generate mice with a mutation in the c-myc proto-oncogene. This mutation is lethal in the homozygous state by 10.5 days of gestation. Of the 28 homozygous embryos analyzed, 86% showed evidence of cardiac failure. Since the c-myc phenotype has other abnormalities in addition to cardiac malfunction, the first goal of this proposal is to determine whether the cardiac defect is due to the c-myc mutation in cells destined to form the heart. To this end, rescue of c-myc mutant mice will be attempted with a c-myc transgene which is expressed in cardiac tissue only. Development can be viewed as a balance between proliferation and differentiation, and in vivo evidence suggests a role for c-myc in that relationship. The failure of cardiac development may be due to a lack of division of myocardial cell precursors, failure to differentiate into contractile tissue, or an inability to respond to growth factors associated with cardiac development. Homozygous c-myc mutant embryonic stem cell lines have been generated and will be used to analyze the differentiation capacity of c-myc deficient cells in tissue culture, in teratocarcinomas, and in chimeric embryos. In addition, other potentially hypomorphic alleles will be generated. These will be useful to dissect the relationship between c-myc protein lack of function and phenotype and between myc family members. Specifically homologous recombination in ES cells will be used to replace the c-myc coding sequence with N-myc coding sequences. Comparison with the c-myc null phenotype will allow us to test the ability of N-myc to substitute for c-myc function in a physiological context. In addition, a mutation which allows only the smaller of the two myc translation products to be made will be generated by the hit and run method.